1. Field of the Invention
The invention relates to the determination of elemental composition of substances from ultrahigh resolution mass spectra of the fine structure of isotopic peak patterns.
2. Description of the Related Art
Contemporary instrumentation in mass spectrometry achieves new records in terms of resolving power and mass accuracy of measured substances. This generates new perspectives for analytical sciences enabling new ways for improving the established methods. With increasing resolving power it is possible to apply new methods to determine elemental composition of substances by taking a closer look at resolved isotopic peak clusters.
Mass spectrometric studies always involve the consideration of atomic isotopes in the compounds studied. In the mass spectrometry of organic compounds the atoms carbon, oxygen, nitrogen, sulfur, phosphorus, and hydrogen play the main role. Most of these elements (12C, 14N, 1H, 16O) have one most abundant isotope with around 99% or more abundance, 32S with around 95% abundance, while 31P is the only stable isotope of phosphorus. The remaining isotope(s) of these elements have very minor abundances (e.g. 13C: 1.070%, 2H: 0.0115%, 15N: 0.368%, 18O: 0.205%, 34S: 4.290% and 33S: 0.75%). However, with increasing size of the molecule, i.e. with increasing number of the corresponding atoms, their isotopic peaks—even with minor abundances—start appearing in the spectrum. In the mass spectrum of an organic substance measured in a narrow m/z region covering all detectable isotopic combinations for molecular ions, the monoisotopic peak (MP) corresponds to a molecule composed of the main isotopes 12C, 1H, 14N, 16O, etc. only. Other isotopic combinations appear next to the monoisotopic peak in approximately 1 Da distances (mMP+n with n=1, 2, 3, . . . ). The first of them at the nominal mass mMP+1 is a cluster of peaks that correspond to molecules that contain only one of other than main isotopes of C, H, N, O and S (13C, or only one 2H, or one 15N, or one 17O, or one 33S). The next one with the nominal mass mMP+2 consists of a cluster of peaks corresponding to molecules having either two 13C atoms or two 15N atoms or one 13C plus one 15N, or one 34S, etc. Thus, each of the isotopic peaks at the nominal masses mMP+n consists of a unique multiple-peak system for this substance and, depending on the size of the molecule and on the heteroatoms, it can be quite complex.
Although each one of the isotopic peak clusters at the nominal masses mMP+n consist of multiple-peak systems, in the organic mass spectrometry it became almost customary to refer to the non-monoisotopic peaks as 13C peaks. This is mainly due to the insufficient resolving power of most of the mass spectrometers used for analytical investigations, so that each one of these isotopic clusters at the nominal masses mMP+n appear as one unresolved peak. Therefore, with insufficient resolving powers the information hidden in the fine structure of the isotopic peak clusters cannot be used. The isotopic fine structure only becomes visible when ultrahigh resolution mass spectrometry is used.
Fourier transform ion cyclotron resonance mass spectrometry delivers the highest resolution in all mass spectrometric techniques. New developments in the ICR cells (Nikolaev, E. N.; Boldin, I. A.; Jertz, R.; Baykut, G.: Initial Experimental Characterization of a New Ultra-High Resolution FT-ICR Cell with Dynamic Harmonization; J. Amer. Soc. Mass Spectrom. 2011, 22, 1125-1133; and/or Boldin, I. A.; Nikolaev, E. N.: FT-ICR Cell with Dynamic Harmonization of the Electric Field in the Whole Volume by Shaping of Excitation and Detection Electrode Assembly, Rapid Commun. Mass Spectrom. 2011, 25, 122-126.) allow resolving powers up to tens of millions (at m/z values around 500) in moderate magnetic fields of only 7 Tesla flux density. With these ultrahigh resolving powers, fine structures of isotopic peak clusters can be easily resolved (Nikolaev, E. N.; Jertz, R.; Grigoryev, A.; Baykut, G.: Fine Structure in Isotopic Peak Distributions Measured Using a Dynamically Harmonized Fourier Transform Ion Cyclotron Resonance Cell at 7 T, Anal. Chem. 2012, 84, 2275-2283, incorporated herein by reference in its entirety).
One of the important tasks of mass spectrometry is to determine the elemental composition of a substance. A possible way of getting the elemental composition information is to accurately determine the molecular mass. With increasing mass accuracy the number of possible compositions to be assigned to an investigated substance decreases. Another option is to get help from fragmentation experiments (tandem mass spectrometry, MSn) while evaluating possible and impossible elemental compositions and structures for the substance.
Methods to calculate elemental composition from ultrahigh resolved spectra of isotopic peak clusters do already exist. However, these conventional methods are based on data from acquiring high resolution mass spectra of a complete isotopic pattern, which includes the monoisotopic peak. As mentioned above, the instrumentation used for ultrahigh resolution mass spectra is usually an FT-ICR mass spectrometer or probably also an FT-Orbitrap™ mass spectrometer. Both the ICR-cell (an electromagnetic ion trap) and the Orbitrap™ (an electrostatic ion trap of the Kingdon type) are ion trap type mass spectrometers. In mass spectrometry with trapped ions the resolving power, as well as the mass accuracy tend to decrease if the number of the trapped ions increases. Thus, a drawback of the conventional method for obtaining the elemental composition by acquiring the complete isotopic pattern is that it operates with unnecessarily large number of ions in the measurement cell (ICR cell or Orbitrap™) Consequently, increased space charge and ion-ion interaction phenomena impair the resolving power as well as the mass accuracy. Especially in organic compounds up to molecular weights (MW) of 5,000, the monoisotopic peak is higher than the next isotope (the abundance of 13C is about 1% of 12C). Additionally, in mass spectra of larger organic compounds, e.g., proteins like myoglobin (MW 16 kDa) or bovine serum albumin (MW 66 kDa), the isotopic distribution consists of many more peaks and approaches a Gaussian form having small monoisotopic peaks. Acquiring here the complete spectrum of the isotopic pattern also significantly increases the number of ions in the measurement cell. In other words, while the method to calculate the elemental composition desperately needs the ultrahigh resolution, the experimental part of the method partially destroys the ultrahigh resolution since the way of calculation requires full pattern information. Thus, the acquired fine structure spectra do not appear as highly resolved as they could be.